Evaluation of the lower protein limit in the budding yeast Saccharomyces cerevisiae using TIPI-gTOW

Background: Identifying permissible limits of intracellular parameters such as protein expression provides important information for examining robustness. In this study, we used the TEV protease-mediated induction of protein instability (TIPI) in combination with the genetic Tug-of-War (gTOW) to develop a method to measure the lower limit of protein level. We first tested the feasibility of this method using ADE2 as a marker and then analyzed some cell cycle regulators to reveal genetic interactions.Results: Using TIPI-gTOW, we successfully constructed a strain in which GFP-^TDegFAde2 was expressed at the lower limit, just sufficient to support cellular growth under the -Ade condition by accelerating degradation by TEV protease. We also succeeded in constructing a strain in which the minimal level of GFP-^TDegFCdc20 was expressed by TIPI-gTOW. Using this strain, we studied genetic interactions between cell cycle regulators and CDC20, and the result was highly consistent with the previously identified interactions. Comparison of the experimental data with predictions of a mathematical model revealed some interactions that were not implemented into the current model.Conclusions: TIPI-gTOW is useful for estimating changes in the lower limit of a protein under different conditions, such as different genetic backgrounds and environments. TIPI-gTOW is also useful for analyzing genetic interactions of essential genes whose deletion mutants cannot be obtained.